Factor VIII/vWF is a plasma coagulation complex composed of two differently sized, noncovalently linked, and genetically independent proteins. The higher molecular weight component that is involved in platelet-vessel wall interactions is termed vWF. It is deficient and/or defecrive in the inherited bleeding disorder, von Willebrand,s disease (vWD). The lower molecular weight component, factor VIII-coagulant (VIII:C), is deficient and/or defective in the inherited disorder, hemophilia A. Many acquired disease states can also exhibit low levels of vWF and VIII:C. In contrast, elevated levels of vWF and VIII:C are associated with acute and chronic inflammatory diseases, cancers and thrombotic states.
The process of this invention utilizes antibodies having novel properties in new qualitative and quantitative tests to permit immunologic measurement of vWF. The process is particularly useful in screening assays which may be performed in a general laboratory or clinical setting without the need of expensive equipment or a highly trained staff.
The method will be employed to determine whether humans and other vertebrates are at risk to bleed during surgery or other stress situations caused by inherited or acquired vWD, or for genetically transmitting vWD. It can also be used to measure vWF levels in individuals experiencing or at risk to develop thrombotic states, cancers, and acute and chronic inflammatory disorders.
No simple immunological screening assay for vWF or vWD has heretofore been available, although sophisticated quantitative assays of vWF are performed in large biomedical and commercial laboratories.
vWD is one of the most common inherited bleeding disorders of man and domestic animals Recent estimates of gene frequently of inherited vWD in humans range from 1 in 5000 to as high as 1 in 50 live births of either sex. Within purebred dog families, prevalence can be as high as 70% (Doberman pinschers) and varies between 15% and 45% in many breeds.
vWF is a very large glycoprotein which plays an important role in reactions of platelets with the vessel wall. It is usually deficient in vWD. The laboratory diagnosis of vWD is dependent on demonstrating decreased levels of plasma vWF. When measured immunologically, this property of vWF is termed vWF antigen (vWF:Ag) and will be so identified hereinafter.
Quantitative determination of plasma vWF:Ag by electroimmunoassay (EIA) or radioimmunoassay (RIA) is currently an important tool to determine whether an individual is affected with congenital, inherited or acquired vWD. The Veterinary Hematology Laboratory of the State of New York Department of Health routinely analyzes vWF:Ag levels by EIA of more than 7,500 samples per year of humans, dogs and other species.
The novel process of this invention utilizes a double antibody Enzyme-Linked Immunosorbent Assay (ELISA) to determine vWF:Ag. The process is easy to perform, efficient, accurate and highly sensitive. It is applicable to humans, nonhuman primates, dogs, horses, pigs, mice, rats, guinea pigs, rabbits, cows, and cats and other vertebrates. These animals are valuable as pets, food sources, work animals, zoological exhibits, and for research. No procedure has heretofore been described in which the same antibodies have been successfully employed with such a large number of species.
Physicians and veterinary clinicians have become increasingly aware of the prevalence of vWD as a cause or precipitating factor in abnormal bleeding and consequently the demand for a practical screening test for vWD has arisen. The process of this invention provides such a test.
Silveira et al (Thrombosis Research 43; 91-102, 1986) have described a sandwich ELISA system to measure plasma vWF:Ag in humans. The test utilizes antibodies to human vWF:Ag raised in rabbits. The rabbit antibody is immobilized in the wells of a microtiter plate and acts as the reactive capture surface for vWF:Ag. Serially diluted plasma samples containing either known or unknown levels of vWF:Ag are reacted with the immobilized antibody, and then quantified by detection with a second (sandwich) antibody to human vWF:Ag (goat antihuman vWF:Ag) and a horseradish peroxidase-conjugated porcine antigoat IgG. The peroxidase activity generated by the final antibody is proportional to the captured amount of vWF:Ag and is measured by the color change it catalyses in the substrate orthophenylenediamine (OPD). The color change reaction is subsequently quenched with sulfuric acid and the optical density measured to determine the concentration of vWF:Ag in the plasma tested.
A disadvantage of the above process is that the rabbit and goat antibodies to vWF which function as the initial capture, immobilizing or anchor antibody and the second or sandwich antibody are raised against human vWF:Ag. Because the original source material for preparing human vWF:Ag is human blood, it presents the danger of transmitting blood-borne diseases to other humans. As a result, the test should only be employed in specially equipped and sanitized laboratories designated for handling specimens of human origin (CDC--Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to health-care and public-safety workers. MMWR. 38, (S-6); Jun. 23, 1989, pp. 1-37).
The process of the present invention does not depend upon the utilization of vWF:Ag from humans to raise antibodies. Moreover, and most unexpectedly, the antibodies utilized in the invention are highly species cross-reactive and can be employed to test for vWF:Ag in a wide range of vertebrates, especially mammals.